Despite recent advances in antibiotic therapy and intensive care, Gram-negative bacterial infection and sepsis are widespread problems in critically ill patients. The high mortality of sepsis is in part mediated by bacterial endotoxin, which stimulates macrophages to sequentially release TNF, IFN-gamma, secretory phospholipase A2 (sPLA2), nitric oxide, and HMGB1. Anti-TNF agents are protective against lethal endotoxemia only if given prophylatically;whereas agents capable of inhibiting HMGB1 release or activity can rescue mice from lethal sepsis even when these anti-HMGB1 agents are given after the onset of sepsis. We have demonstrated that, an endogenous ubiquitous polyamine, spermine, accumulates at sites of infection and injury, can attenuate endotoxin-induced HMGB1 release. The spermine-mediated cytokine suppression is dependent on the availability of a negative acute phase protein, fetuin, whose circulating levels are significantly decreased during endotoxemia and sepsis (induced by cecal ligation and puncture, CLP). Notably, intraperitoneal administration of exogenous fetuin 24 hours after CLP significantly rescues mice from lethal sepsis, prompting the necessity to investigate the mechanisms by which spermine and/or fetuin attenuate endotoxin-induced HMGB1 release, and confer protection against lethal endotoxemia and sepsis. In light of emerging evidence implicating a potential role for JNK MAP kinase, sPLA2, and inducible nitric oxide synthase (iNOS) in bacterial endotoxin-induced HMGB1 release, we will first determine whether spermine and/or fetuin inhibit endotoxin-induced HMGB1 release through a JNK MAP kinase, sPLA2, or iNOS-dependent mechanism (Specific Aim 1). We will then determine whether administration of exogenous fetuin and/or spermine, or genetic disruption of fetuin expression, influences animal survival in endotoxemia and sepsis (Specific Aim 2). Lastly, we will delineate the specific mechanisms of spermine- and/or fetuin- mediated protection by determining whether administration of spermine and/or fetuin alters systemic accumulation of pro-inflammatory cytokines, or alternatively influences clearance of invading pathogens and/or apoptotic cells in wild-type or mutant mice deficient in TNF, IFN-gamma, or fetuin (Specific Aim 3). Answers to these questions will improve our understanding of the pathophysiology of sepsis, and shed light on the development of novel therapeutic strategies for treatment of lethal systemic inflammatory diseases.